Inhibitory effect of curcumin on malignant biological behavior and Wnt/β-catenin pathway of uveal melanoma cells

Objective

To explore the inhibitory effect of curcumin on the malignant biological behavior of uveal melanoma (UM) and its possible mechanism.

Methods

M23 cells were cultured in curcumin medium with different concentrations (0, 20, 40 and 80 μmol/L) for 48 hours, respectively.The morphological changes of cells were observed under an inverted microscope.The cell survival rate was detected by the cell counting kit-8 (CCK-8) method.The apoptosis, colony formation, migration and invasion of cells were detected by flow cytometry, plate clone formation experiment, cell scratch experiment and Transwell assay, respectively.The relative expressions of genes related to Wnt/β-catenin pathway, c-Myc, Cyclin D1, Survivin and matrix metallo proteinase 9 (MMP-9) mRNA in cells were detected by real-time fluorescence quantitative PCR.The relative expressions of proteins related to Wnt/β-catenin pathway, c-Myc, Cyclin D1, Survivin, MMP-9 and β-catenin, glycogen synthase kinase 3β (GSK-3β), phosphorylated GSK-3β (p-GSK-3β) and axis inhibition protein 2 (Axin2) proteins were detected by Western blot.Another 20 female BALB/c mice were selected and injected with M23 cell suspension under the subcutaneous fat pad in the left posterior abdomen to establish the in vivo M23 transplanted tumor model.The mice successfully modeled were randomly divided into model group, low-dose curcumin group, medium-dose curcumin group and high-dose curcumin group according to the random number table method, which was intraperitoneally injected with 0, 10, 20 and 40 mg/kg curcumin physiological saline solution respectively.After a continuous injection for 30 days, the subcutaneous tumor was stripped and weighed.The animal experiment process followed the 3Rs principle of animal research and was approved by the Laboratory Animal Ethics Committee of Inner Mongolia Baotou Steel Hospital (No.2021MER-023).

Results

The cell survival rate, the number of colony formation, the apoptosis rate, the cell invasion rate and the cell migration rate were (100.00±0.00)%, 128.67±9.18, (1.33±0.29)%, (89.76±4.57)% and 148.33±8.18 in 0 μmol/L curcumin group, (83.78±4.59)%, 100.33±8.73, (14.53±2.04)%, (65.43±3.70)% and 125.33±7.41 in 20 μmol/L curcumin group, (66.09±3.92)%, 58.67±6.55, (27.23±3.56)%, (34.83±2.19)% and 73.67±6.34 in 40 μmol/L curcumin group, and (47.16±3.63)%, 31.67±4.92, (44.73±4.36)%, (18.82±1.99)% and 45.67±5.31 in 80 μmol/L curcumin group.There were statistically significant differences in the survival rate, colony formation number, cell apoptosis rate, migration rate and invasion rate of M23 cells among the four groups (F=125.321, 97.941, 72.516, 277.097, 139.006; all at P<0.001). With the increase of curcumin concentration, the cell survival rate, colony formation number, cell migration rate and cell invasion number decreased obviously, and the cell apoptosis rate increased obviously, and the pairwise comparisons showed significant differences (all at P<0.05). With the increase of curcumin concentration, the relative expression levels of c-Myc, Cyclin D1, Survivin, MMP-9 mRNA and proteins, β-catenin and p-GSK-3β proteins decreased significantly, while the relative expression level of Axin2 protein increased significantly, showing significant differences in pairwise comparisons (all at P<0.05). The tumor tissue weight of mice decreased with the increase of curcumin dosage, and the pairwise comparisons were statistically significant (all at P<0.05).

Conclusions

Curcumin can inhibit the proliferation, migration, invasion and other malignant biological behaviors of UM M23 cells, inhibit tumor growth and promote cell apoptosis.Its mechanism may be related to blocking the activation of Wnt/β-catenin pathway.

Curcumin; Uveal melanoma; Biological behavior; Wnt/β-catenin pathway